Interactions between NFκB and Its Inhibitor iκB: Biophysical Characterization of a NFκB/iκB-α Complex

The N-terminal domain (1–318 amino acids) of mouse NFB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length iB- (MAD3, 1–317 amino acids) molecule was generated by binding the E. coli-derived iB- to the purified NFB and purifying the complex by sequential chromatography. The stoichiometry of NFB to iB in the complex was determined to be 2 to 1 by light scattering and SDS–polyacrylamide gel electrophoresis. The secondary structure of the NFB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFB occurs upon binding of DNA. The FTIR spectrum of the NFB/iB complex indicates that its secondary structure is composed of 17% -helix, 39% -strand, 18% irregular structures, and 26% -turns and loops. By comparing these data to the FTIR data for NFB alone, it is concluded that the iB (MAD3) in the complex contains 35% -helix, 27% -strand, 22% irregular structures, and 16% -turns and loops. Circular dichroism (CD) analysis of a shorter form of iB (pp40) indicates that it contains at least 20% -helix and that the iB subunit accounts for nearly all of the -helix present in the NFB/iB complex, consistent with the FTIR results. The stabilities of NFB, iB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of iB is enhanced upon the formation of the NFB/iB complex.     

The IκB kinase complex in NF-κB regulation and beyond

The IκB kinase (IKK) complex is the signal integration hub for NF-κB activation. Composed of two serine-threonine kinases (IKKα and IKKβ) and the regulatory subunit NEMO (also known as IKKγ), the IKK complex integrates signals from all NF-κB activating stimuli to catalyze the phosphorylation of various IκB and NF-κB proteins, as well as of other substrates. Since the discovery of the IKK complex components about 15 years ago, tremendous progress has been made in the understanding of the IKK architecture and its integration into signaling networks. In addition to the control of NF-κB, IKK subunits mediate the crosstalk with other pathways, thereby extending the complexity of their biological function. This review summarizes recent advances in IKK biology and focuses on emerging aspects of IKK structure, regulation and function.     

核因子κB研究进展

核因子κB(nuclear factor κB,NF-κB)是一种广泛存在于各种细胞、具有多种调节作用的转录因子。它在正常情况下在胞浆内与抑制蛋白(IκB)结合而呈非活性状态。当细胞受到各种刺激原如紫外辐射、细胞因子(如TNF-α、IL-1)、活性氧作用时,NF-κB与IκB解离并进入细胞核内,与特定的启动子结合,从而调控各种基因的表达,如细胞因子、炎症因子、黏附分子等。NF-κB在炎症发生时复杂的细胞因子网络中起着中心调节作用。在细胞增殖、分化和凋亡及肿瘤发生中NF-κB也扮演着重要角色。以NF-κB作为药物作用的靶点,通过调节NF-κB的活性,可改善某些疾病的治疗效果。     

Lipopolysaccharide-Induced NF-κB Activation in Human Endothelial Cells Involves Degradation of IκBα but Not IκBβ

We studied the signal transduction pathways involved in NF-κB activation and the induction of the cytoprotective A20 gene by lipopolysaccharide (LPS) in human umbilical vein endothelial cells (HUVEC). LPS induced human A20 mRNA expression with a maximum level 2 h after stimulation. The proteasome inhibitorN-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) and the tyrosine kinase inhibitor herbimycin A (HMA) blocked A20 mRNA expression and partially inhibited NF-κB DNA-binding activity induced by LPS treatment. LPS induced IκBα degradation at 30–60 min after treatment, but did not induce IκBβ degradation up to 120 min. In contrast, TNF-α rapidly induced IκBα degradation within 5 min and IκBβ degradation within 15 min. Cycloheximide did not prevent LPS-induced IκBα degradation, indicating that newly synthesized proteins induced by LPS were not involved in LPS-stimulated IκBα degradation. LPS-induced IκBα degradation was inhibited by ALLN, confirming that ALLN inhibits NF-κB activation by preventing IκBα degradation. Of note, HMA also inhibited LPS-induced IκBα degradation. However, tyrosine phosphorylation of IκBα itself was not elicited by LPS stimulation, suggesting that tyrosine phosphorylation of a protein(s) upstream of IκBα is required for subsequent degradation. We conclude that in HUVEC, LPS induces NF-κB-dependent genes through degradation of IκBα, not IκBβ, and propose that this degradation is induced in part by HMA-sensitive kinase(s) upstream of IκBα.     

Rel／NF—κB／IκB／IKK信号转导途径研究进展

真核细胞枋转导因子Ｒｅｌ／ＮＦ－κＢ家族广泛调控着自昆虫至人类的免疫和炎症反应中一系列基因的表达。静息状态下,ＮＦ－κＢ二聚体与抑制性蛋白ＩκＢ结合而存在于胞质中,当细胞受外源性刺激哩,ＮＦ－κＢ活化进入核内发挥其功能。目前,外源性信号活化ＮＦ－κＢ的机制已初步阐明,Ｒｅｌ／ＮＦ－κＢ／ＩκＢ／ＩＫＫ信号转导途径在蛋白质水平的相互调控,以及在肿瘤发生中的意义的研究也已获得一定进展。本文综述了近年来     

IκB激酶的研究进展

ＩκＢ激酶的研究进展陈红清（中国医学科学院、中国协和医科大学皮肤病研究所,南京２１００４２关键词ＩκＢ激酶信号转导转录因子中κ基因结合核因子（ＮＦ－κＢ）由属于癌基因ｒｅｌ表达的Ｒｅｌ蛋白家族的２个亚基组成,最常见的形式是由ｐ６５（Ｒｅｌ－Ａ）和ｐ５...     

1,6-O,O-diacetylbritannilactones inhibits IκB kinase β-dependent NF-κB activation

To determine the chemical constituents responsible for pharmacological effects of Inula britannica-F., three specific sesquiterpene lactones in Inula britannica were isolated from chloroform extract and identified, including britannilactone (BL), 1-O-acetylbritannilactone (ABLO), and 1,6-O,O-diacetylbritannilactone (ABLOO). Electrophoretic mobility shift assay (EMSA) was performed to detect the nuclear translocation of nuclear factor-κB (NF-κB) p65. The expressions of IκBα, pIκBα, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IκB kinase α/β (IKKα/β) and NF-κB kinase (NIK) were detected by Western blot and RT-PCR. We found that acetyl side groups enhanced the inhibitory action of the agents on LPS/IFN-γ-induced iNOS and COX-2 expression. Their inhibiting activity was positive correlation with the acetyl side group number. The effects of LPS/IFN-γ were reversed by ABLOO, and BL without acetyl side groups showed only a weak inhibitory action. Further study indicated that ABLOO markedly inhibited the phosphorylation of IKKβ down to based level, but not IKKα, corresponding with decreased in IκBα degradation and phosphorylation induced by LPS/IFN-γ, resulting in the suppression of NF-κB nuclear translocation and activity. These results suggest that the acetyl moieties add to the lipophilicity, and consequently enhance cellular penetration, so that ABLOO possess the most anti-inflammatory effect and may be a potent lead structure for the development of therapeutic and cytokine-suppressing remedies valuable for the treatment of various inflammatory diseases.     

IκB激酶的激活及其在NF-κB活化过程中的作用

在NF-κB二聚体活化过程中,IκB激酶（IKK）通过对抑制性蛋白κB(IκBs)的磷酸化而扮演关键的角色.IKK复合物在胞浆内有多种存在形式,其中,IKK-α、IKK-β两者氨基酸序列52%的同源性,空间构象相似,常为催化亚单位,而IKK-γ则为调节亚单位,它们以不同的方式活化IκBs.核因子κB诱导激酶（NIK）与丝裂原活化蛋白激酶激酶激酶-1（MEKK₁）均为IKK的上游激酶,NIK可引起IKK-α Ser176、IKK-β相应位点的磷酸化,而MEKK₁主要引起IKK-β的活化.通过级联反应,使IκBs磷酸化而与NF-κB解离,致使NF-κB被激活并易位入核,启动免疫及炎症相关的基因转录.     

Non-canonical NF-κB signaling pathway

The non-canonical NF-κB pathway is an important arm of NF-κB signaling that predominantly targets activation of the p52/RelB NF-κB complex. This pathway depends on the inducible processing of p100, a molecule functioning as both the precursor of p52 and a RelB-specific inhibitor. A central signaling component of the non-canonical pathway is NF-κB-inducing kinase (NIK), which integrates signals from a subset of TNF receptor family members and activates a downstream kinase, IκB kinase-α (IKKα), for triggering p100 phosphorylation and processing. A unique mechanism of NIK regulation is through its fate control: the basal level of NIK is kept low by a TRAF-cIAP destruction complex and signal-induced non-canonical NF-κB signaling involves NIK stabilization. Tight control of the fate of NIK is important, since deregulated NIK accumulation is associated with lymphoid malignancies.     

NF-κB的生物学功能

NF—κB因其广泛参与机体的免疫及其它应激反应而受到人们的关注,其常见的形式是由p50和p65组成的异源二聚体。细胞受到外部因素刺激后,NF-κB由细胞质转移到细胞核中,并发生磷酸化和乙酰化,启动相关基因的表达。目前研究表明受NF-κB调节的基因有200多种,其激活因子不少于150种,所以对NF-κB在各种条件下的激活过程及信号传导网络的研究具有重要的意义。本文综合了当前关于NF—κB研究的最新进展,着重阐述了由TNF-α、IL-Ⅰ及LPS刺激而引起NF—κB激活的信号传导通路,并进一步阐述了其在人类某些疾病当中的生物学功能。     

NF—κB与疾病

NF－κB是一种多极性基因调控性能的转录因子,能够激活若干个炎症反应、机体免疫反应及多种细胞因子的基因转录过程,从而控制它们的生物合成。本文综述了近年来国外对NF－κB的研究进展,对NF－κB的结构组成、激活途径、生物学功能及其与多种疾病的关系作一介绍。     

Tetrandrine suppresses LPS-induced astrocyte activation via modulating IKKs-IκBα-NF-κB signaling pathway

Astrocyte activation has been implicated in the pathogenesis of many neurological diseases. These reactive astrocytes are capable of producing a variety of proinflammatory mediators and potentially neurotoxic compounds, such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1beta (IL-1beta). In this study, we examined the suppressive effects of Tetrandrine (TET) on astrocyte activation induced by lipopolysaccharide (LPS) in vitro. We found that TET decreased the release of NO, TNF-alpha, IL-6 and IL-1beta in LPS-activated astrocytes. Also mRNA expression levels of inducible nitric oxide synthase (iNOS), macrophage inflammatory protein-1alpha (MIP-1alpha) and vascular cell adhesion molecule-1 (VCAM-1) were inhibited in TET pretreated astrocytes. Such suppressive effects might be resulted from the inhibition of nuclear factor kappa B (NF-kappaB) activation through downregulating IkappaB kinases (IKKs) phosphoration, which decreased inhibitor of nuclear factor-kappaB-alpha (IkappaBalpha) phosphoration and degradation. Our results suggest that TET acted to regulate astrocyte activation through inhibiting IKKs-IkappaBalpha-NF-kappaB signaling pathway.